Use of decorine for increasing muscle mass

ABSTRACT

The invention concerns decorin for increasing muscle mass, particularly in the treatment of muscular dystrophies.

TECHNICAL DOMAIN

The aim of this invention is to increase muscle mass in humans or animals.

More specifically, it advocates the use of decorin to develop muscle mass, particularly for treating pathological conditions associated with muscular wasting, such as muscular dystrophy.

PRIOR STATE OF THE ART

Neuromuscular diseases include various conditions that are generally associated with temporary or permanent loss of muscular strength. This loss of strength is usually accompanied by muscular wasting, also known as amyotrophia.

Myopathies, which involve damage to the actual muscle fibres, are an important group of these muscular diseases, and among them, progressive muscular dystrophies are characterised by a decrease in muscular strength, generally with atrophy of the muscles, as well as abnormalities in the muscle biopsy showing modifications of the tissue. This group notably includes Duchenne muscular dystrophy (or DMD), Becker muscular dystrophy (or BMD) and the limb girdle muscular dystrophies.

Associated genetic abnormalities have been identified for some of these diseases. Duchenne or Becker muscular dystrophies are related to alterations in the gene encoding dystrophin, type 2A limb girdle muscular dystrophy (LGMD 2A or calpainopathy) to alterations in the calpain 3 gene, while the sarcoglycanopathies or the dystrophy types LGMD 2C, LGMD 2D, LGMD 2E, LGMD 2F are related to defects in the γ-, α-, β- and δ-sarcoglycan genes respectively (McNally E M, Pytel P, Muscle diseases: the muscular dystrophies. Annu Rev Pathol. 2007; Vol 2: 87-109).

In these particular cases, different gene therapy strategies are being developed but are difficult to put into practice.

Nevertheless and more generally in all cases of muscular wasting, there is a clear need to develop technical solutions to increase muscle mass and/or volume.

The document WO 2005/094446 identified antibodies against an epitope located between residues 40 and 64 of mature human myostatin which could increase muscle mass. However, this strategy based on the recognition of myostatin by an antibody is not free of problems. Alternative solutions therefore need to be found.

The present invention is based on the discovery by the inventors of this property of decorin.

Decorin belongs to the SLRP (Small Leucine-Rich Proteoglycan) family of proteins and includes an LRR (Leucine-Rich Repeat) segment. Decorin is a member of class I of the SLRPs. The members of this family are secreted with a propeptide which, in some cases, is cleaved. Decorin also has a glycosaminoglycan (GAG) chain.

Decorin is a protein of the extracellular matrix, with a similar structure to that of the protein biglycan. It plays a role in assembling the matrix and interacts with various partners, such as type I, II, III and IV collagen, or TGF-beta (Ameye L, Young M F, Mice deficient in small leucine-rich proteoglycans: novel in vivo models for osteoporosis, osteoarthritis, Ehlers-Danlos syndrome, muscular dystrophy, and corneal diseases. Glycobiology 2002; Vol 12:107 R-116R; Reed C C, Iozzo R V, The role of decorin in collagen fibrillogenesis and skin homeostasis. Glycoconj J. 2002; Vol 19(4-5): 249-55).

On the basis of its interaction with TGF-beta, WO 96/25178 proposed the use of decorin to treat diseases associated with tissue fibrosis, i.e. excessive production of extracellular matrix, without relating this to the muscle mass problem.

DETAILED DESCRIPTION OF THE INVENTION

The present invention thus concerns the use of decorin to counter muscle wasting and even to increase muscle mass.

For this invention, the term “muscle mass” could be replaced either by muscle weight or volume.

More precisely the invention also concerns:

-   -   a composition containing decorin to treat diseases associated         with muscle wasting;     -   the use of decorin in the preparation of a medicinal product for         treating diseases associated with muscle wasting;     -   a composition containing decorin to increase muscle mass;     -   the use of decorin to increase muscle mass.

There are a number of conditions in which muscle wasting occurs.

Firstly, it may result from pathological conditions, particularly in the case of neuromuscular diseases. Duchenne muscular dystrophy is a disease particularly targeted, but all forms of neuromuscular diseases, especially muscular dystrophies, can be treated.

In addition, cachexia or marasmus is also a medical condition targeted by this invention. This state is characterized by extreme thinness, especially of the muscles, caused by prolonged illness or inadequate calorie or protein intake.

This condition is particularly seen in cases of chronic diseases such as cancer or AIDS or in individuals with either heart failure, where there is atrophy of skeletal muscles in 68% of patients, or urinary incontinence.

Although not actually considered as pathological, some situations are associated with muscle wasting: ageing, prolonged immobilisation etc. Here again, therefore, there is a reason for increasing the muscle mass.

The invention also offers the possibility particularly in the area of food production of increasing animal meat production. The use of decorin is therefore of particular interest in animals.

This invention is based therefore on detection of the stimulating properties of decorin, particularly related to muscle volume.

In the invention, “decorin” is used generically to mean the protein described by Krusius et al. (Krusius T., Ruoslahti E., Primary structure of an extracellular matrix proteoglycan core protein deduced from cloned cDNA. Proc Natl Acad Sci USA 1986; Vol 83(20): 7683-87). The human protein described in this document has the sequence SEQ ID NO: 1. It is in the form of a preproprotein of 359 amino acids. Both native proteins and those deprived of their propeptide and/or their signal sequence (329 aa), are covered by this invention.

Although decorin naturally has a glycosaminoglycan (GAG) chain, a decorin without GAG (GAG−) can also be used in the context of this invention. This can, for example, be obtained by enzyme treatment.

The decorin can be obtained from any organism, but in this invention, decorin of human origin is preferred. More generally and advantageously, the protein comes from the same organism as the organism into which it will be administered. Preferably therefore, for therapeutic indications in humans, human decorin is used to advantage.

One of the primary benefits indeed of the solution proposed in this invention is that decorin is a protein naturally present in mammals, especially humans, and therefore a priori is not likely to cause side effects or immune responses.

It has also been shown for human decorin that transcriptional variants exist (variants b, c, d and e), resulting in protein isoforms, of sequence SEQ ID NO: 2, 3, 4 and 5, respectively, included in this submission.

In the context of the invention, the term “decorin” thus has a wide meaning and covers:

-   -   the native protein, particularly the sequence SEQ ID NO: 1;     -   the protein with or without the GAG chain (GAG+ or GAG−,         respectively);     -   the protein lacking the propeptide and/or the signal sequence;     -   variants of these proteins, especially embodied in the sequences         SEQ ID NOS: 2 to 5;     -   more generally active fragments of these proteins,     -   or active derivatives or functional equivalents.

As far as the fragments or derivatives are concerned, they are, of course, active fragments or derivatives. The activity in question which these fragments or derivatives must possess concerns the ability to increase muscle mass, which is easily assessed by using the test described in this submission.

In practice, they are to advantage 60% identical to one of sequences SEQ ID NO: 1 to 7, even more advantageously 70%, 80% or 90% identical.

Thus, by way of example for the derivatives, it could be sequence SEQ ID NO: 6 corresponding to the murine protein of 354 aa, which is 80% identical to the human sequence SEQ ID NO: 1.

According to a preferred embodiment of the invention, the decorin is in the form of an active fragment. To advantage by “fragment” we mean a peptide containing less than 100 amino acids, to even greater advantage, less than 50 amino acids.

The use of a peptide instead of the protein has certain advantages, particularly in terms of its production but also concerning the possible risk of undesirable interference in vivo.

It has been shown as part of this application that a 41 residue fragment of the N-terminal part of murine decorin (SEQ ID NO: 7) corresponding to residues 31-71 of the sequence SEQ ID NO: 6, reported to fix zinc (Yang V W, LaBrenz S R, Rosenberg L C, McQuillan D, Höök M. Decorin is a Zn2+metalloprotein. J Biol Chem. 1999, 274(18): 12454-60), had the required activity. It has also been shown that an even smaller fragment of 30 residues (residues 42 to 71 of the sequence SEQ ID NO: 6 corresponding to SEQ ID NO: 15) was also active.

The corresponding domain, present in human decorin, can be easily determined by the methodology described in this document. Such a fragment may for example have the sequence SEQ ID NO: 16.

More generally, the invention therefore concerns the use of a fragment of decorin including the zinc binding domain, in practice the residues 31 to 71, possibly 42 to 71 of the murine sequence. In a particular embodiment, the sequence of the fragment in question corresponds to sequence SEQ ID NO: 7 or SEQ ID NO: 15. In addition, fragments which are to advantage 50% identical to SEQ ID NO: 7 or 15, or even more advantageously 60%, 70%, 80% or 90% identical to them, and which retain their ability to bind zinc, are also covered by this invention.

In addition, decorin, its fragments and active derivatives may also be in the form of fusion proteins or chimeric proteins with another protein fragment at their N- or C-terminal ends, which can, for example, but without being limited to this, increase the residence time of the protein in the organism. A preferred example is the chimera consisting of the constant region of mammalian IgGs, attached via a hinge sequence to decorin or one of its fragments. Another example is human or mammalian albumin, also attached to decorin or to a protein fragment of decorin. Such combinations can be obtained both from a recombinant cDNA and by chemical bonding of the 2 proteins.

The present invention is therefore based on an exogenous supply of decorin. In fact, the composition covered by the invention consists of either the protein as such or a system producing the protein.

As far as the protein itself is concerned, it could be either native decorin, purified from an organism naturally producing this protein, or a recombinant protein produced by any of the synthesis systems available and known to those working in the field.

Alternatively, a nucleic acid sequence encoding decorin is put into an expression system, to advantage under the control of a promoter in a vector. After introduction into the body, the decorin is produced in vivo. The transfer of the nucleic acids (DNA or RNA) can be done either with viral approaches to gene transfer (e.g. adeno-associated virus or AAV) or with non-viral approaches (e.g. by simple intramuscular injection of a plasmid). Genomic DNA may be of interest since in some cases, the presence of introns stabilises the prespliced mRNA and improves its stability in the nucleus and its export, which leads to better protein expression.

Decorin, its derivatives or fragments, can thus be provided in the form of nucleic acids, particularly DNA or RNA, and may for example be in the form of transcripts occurring naturally in humans or the mouse. The following sequences are preferred:

-   -   Sequence SEQ ID NO: 8, corresponding to the A1 variant         (Accession Number NM_(—)001920.3), which is the longest         transcript and encodes the isoform a of the human decorin         sequence SEQ ID NO: 1 (Accession Number NP_(—)001911);     -   Sequence SEQ ID NO: 9, corresponding to the A2 variant         (Accession Number NM_(—)133503.2), which uses an alternative         exon at the 5′UTR compared with the variant A1 and encodes the         same protein sequence SEQ ID NO: 1 (Accession Number         NP_(—)598010.1);     -   Sequence SEQ ID NO: 10, corresponding to the B variant         (Accession Number NM_(—)133504.2), which lacks exons 3 and 4 in         the coding region, compared with the A1 variant. This causes no         change in reading frame but codes for an isoform b of the         protein, which lacks an internal fragment of 109 aa, and has the         sequence SEQ ID NO: 2 (Accession Number NP_(—)598011.1);     -   Sequence SEQ ID NO: 11, corresponding to the C variant         (Accession Number NM_(—)133505.2), which lacks exons 3, 4 and 5         in the coding region, compared with the A1 variant. This causes         a change of internal reading frame and the isoform c encoded of         SEQ ID NO: 3 (Accession Number NP_(—)598012.1) is shorter than         isoform a by 147 amino acids;     -   Sequence SEQ ID NO: 12, corresponding to the D variant         (Accession Number NM_(—)133506.2), which lacks exons 4, 5, 6 and         7 in the coding region, compared with the A1 variant. This         causes no change in reading frame but codes for an isoform d of         the protein, which lacks an internal fragment of 187 aa, and has         the sequence SEQ ID NO: 4 (Accession Number NP_(—)598013.1);     -   Sequence SEQ ID NO: 13, corresponding to the E variant         (Accession Number NM_(—)133507.2), which lacks exons 3, 4, 5, 6         and 7 in the coding region, compared with the A1 variant. This         causes a change of internal reading frame and the isoform e         encoded of SEQ ID NO: 5 (Accession Number NP_(—)598014.1) is         shorter than isoform a by 284 amino acids;     -   Sequence SEQ ID NO: 14, encoding the murine protein sequence SEQ         ID NO: 6 (Accession Number P28654).

As already mentioned, decorin is known to be a zinc metalloprotein. Owing to this and in order to potentiate its activity, one could choose to provide additional zinc to that naturally available in the organism to which the decorin is administered. Thus, according to this embodiment, the composition containing the decorin also includes zinc, e.g. as zinc chloride, preferably at a concentration between 1 and 50 μM, even equal to 15 μM.

A composition containing decorin according to the invention for the treatment of diseases associated with muscle wasting or intended to increase muscle mass may also contain any acceptable compound or excipient, particularly a pharmaceutical compound or excipient. The route of administration may be intramuscular or intravenous, or even subcutaneous, intraperitoneal or oral.

To promote the engraftment of precursor cells or stem cells, it may be advantageous to combine the administration of decorin with the cell grafts (myoblasts, stem cells etc.). This administration can be simultaneous or separated in time.

It can also be advantageous to combine gene therapy for the treatment of a neuromuscular disease with administration of decorin. In a preferred embodiment, a therapeutic gene is associated with decorin treatment. Administration of the two treatments can be simultaneous or separated in time.

The beneficial effects of decorin result in an increase in muscle volume (either mass or weight), due to an increase in the area of fibres possibly associated with an increase in the number of fibres. These positive effects can be observed in the various different skeletal muscles, both in an organism with a disease affecting its muscle mass and in a healthy individual. In principle, there are no side effects and no immune reaction.

EXAMPLES OF EMBODIMENTS

The invention and the advantages resulting from it are better illustrated by the following examples of embodiments and the attached figures. These are in no way limiting.

The invention is further illustrated by means of recombinant mouse decorin injected intramuscularly into mdx mice with a gene encoding an altered dystrophin and serving as an experimental model of Duchenne muscular dystrophy, and gamma-sarcoglycan−/−mice (mouse model of sarcoglycanopathies on a pure C57/B16 background).

LEGENDS TO THE FIGURES

FIG. 1 is a view of the tibialis anterior muscle taken from mdx mouse 7 that had received (on the left) or not (on the right) an intramuscular injection of decorin.

FIG. 2 is a view of the tibialis anterior muscle taken from mdx mouse 8 that had received (on the left) or not (on the right) an intramuscular injection of decorin.

FIG. 3 is a view of the tibialis anterior muscle taken from gamma-sarcoglycan−/−mouse 4 at D18 that had received (on the left) or not (on the right) an intramuscular injection of decorin.

FIG. 4 is a view of a cross-section of the tibialis anterior muscle taken from gamma-sarcoglycan−/−mouse 4 at D18 that had received (LTA4 on the right of the figure) or not (RTA4 on the left of the figure) an intramuscular injection of decorin.

I) MATERIALS AND METHODS

Preparing the mDecorin Solution

The protein used was recombinant mouse decorin (mDecorin) of sequence SEQ ID NO: 6, provided by R&D Systems.

Twenty-four to forty hours before the injection, 100 μl of 150 mM sterile NaCl and 6 μl of 250 μM ZnCl₂ were added to 100 μg of mDecorin powder. The final volume was 106 μl with a final concentration of approximately 1 μg/μl. For the injections into the control muscles a mixture was also prepared of 100 μl of 150 mM NaCl and 6 μl of 250 μM ZnCl₂. All these solutions, after being vortexed, were stored at 4° C.

In Vivo Injection

All the mice were treated according to EU directives on human health and the use of experimental animals.

mdx dystrophic (S-linked muscular dystrophy) or gamma-sarcoglycan−/−mice were used that were at least 6 weeks old. 20 μg of mDecorin, i.e. 22 μl of the solution described above (20 μg Decorin+15 μM ZnCl₂/22 μl NaCl), were injected into the left tibialis anterior (LTA), the muscle treated. 22 μl of the control solution described above (15 μM ZnCl₂/22 μl NaCl) were administered into the control muscle, the right tibialis anterior (RTA). A specific number of days after injection, the mice were sacrificed and the RTA and LTA were removed, weighed then frozen for further histological study.

Preparation and Injection of the Solution Containing the Peptide mDCN 31-71:

The peptide with the sequence SEQ ID NO: 7 was synthesised by the company NeoMPS with purity >65%. It was dissolved at 2 mg/ml in 150 mM NaCl and stored at −80° C.

For injections, the preparation protocol was identical to that used for the protein, i.e. 24-40 hours before injection, the desired amount of peptide was removed from the stock solution and mixed with a solution of zinc chloride (ZnCl₂) and 150 mM NaCl, to produce a final zinc concentration of 15 μM. The injection protocol was identical to that used for the protein.

Histological Analyses

Laminin Labelling:

Cryostat sections (8 μm) were made of treated and control muscles using standard techniques. The slides were fixed with Dakopen (DAKO®, ref.: S 2002) for 10 minutes open to the air and then blocked with a solution of PBS/10% goat serum for 30 min at room temperature in a humidity chamber. The rabbit anti-laminin antibody (DAKO®, ref.: Z0097) was applied to the slides at a dilution of 1:1000 for 12 hours in the humidity chamber. The slides were then rinsed in PSB (5 minutes) while being agitated and the secondary antibody (Envision HRP rabbit kit) was applied to the slides in a humidity chamber for 30 min at room temperature. After rinsing the slides in PBS (5 minutes) while being agitated, the DAB (DAKO®, ref.: K 3466) was applied to the sections for 2 to 5 minutes at room temperature in a humidity chamber. The slides were rinsed constantly and were mounted in the fume cupboard. The results were analysed using ELLIX software.

HPS Staining:

Cryostat sections (8 μm) were made of treated and control muscles using standard techniques. The slides were immersed in Harris haematoxylin for 3 minutes and then rinsed with running water. The slides were then put into acid alcohol, rinsed and soaked in Scott's tap water substitute for one minute. After rinsing, the slides were immersed in phloxine for 30 seconds, rinsed with running water and soaked in absolute alcohol for one minute. After exposure to the saffron for 3 minutes, the slides were rinsed with absolute alcohol and mounted with Eukitt resin, the solvent for which is xylene. The results were analysed using the CARTHOGRAPH program.

II) RESULTS

1/ Weight of Muscles at Different Times after Injection into Dystrophic mdx Mice:

The RTA and LTA muscles were collected 7 (D7), 14 (D14) or 21 (D21) days after the injection and weighed. The experiment was repeated on three separate mice each time. The results are summarised in the following tables:

Day 7:

Growth in % Mouse Muscles Weight (g) (100*LTA/RTA) − 100 Mouse 1 RTA 1 0.0661 3.18 LTA 1 0.0682 Mouse 2 RTA 2 0.0774 0.90 LTA 2 0.0781 Mouse 3 RTA 3 0.0749 2.94 LTA 3 0.0771

Day 14:

Mouse Muscles Weight Growth Mouse 4 RTA 4 0.0707 58.98 LTA 4 0.1124 Mouse 5 RTA 5 0.0694 48.41 LTA 5 0.103 Mouse 6 RTA 6 0.0854 6.67 LTA 6 0.0911

Day 21:

Mouse Muscles Weight Growth Mouse 7 RTA 7 0.068 53.09 LTA 7 0.1041 Mouse 8 RTA 8 0.0567 66.31 LTA 8 0.0943 Mouse 9 RTA 9 0.0731 37.21 LTA 9 0.1003

The difference in muscle mass at day 21 between an mdx mouse that had received or had not received an intramuscular injection of decorin can be seen in FIGS. 1 and 2 for mice 7 and 8, respectively. There is a clear increase in muscle mass (+53.09% and +66.31%, respectively).

2/ Weight of Muscles at D18 after Injection into Dystrophic Gamma-Sarcoglycan−/−Mice:

A second series of experiments was performed on four gamma-sarcoglycan−/−mice. The protocol was identical to that described for mdx mice. The mice were sacrificed on D18. The results, shown in FIGS. 3 and 4, are presented in the following table:

Mouse Muscles Weight (g) Growth Mouse 1 RTA 1 0.0456 10.75 LTA 1 0.0505 Mouse 2 RTA 2 0.0413 17.43 LTA 2 0.0485 Mouse 3 RTA 3 0.0528 12.31 LTA 3 0.0593 Mouse 4 RTA 4 0.0444 24.10 LTA 4 0.0551 3/ Injection of the Peptide 31-71 Derived from the N-Terminal Part of Murine Decorin in mdx Mice:

To verify whether the N-terminal part of decorin is sufficient to produce observable increases in muscle mass, similar experiments were performed in the presence of the mDCN 31-71 peptide (SEQ ID NO: 7) corresponding to residues 31-71 of murine decorin (SEQ ID NO:6). This peptide has been described as being sufficient and necessary for binding zinc (Yang V W, LaBrenz S R, Rosenberg L C, McQuillan D, Höök M. Decorin is a Zn2+ metalloprotein. J Biol Chem. 1999, 274(18): 12454-60.).

mdx mice were injected intramuscularly into the TA with the following formulations:

LTA 1: 65 μg peptide 41 DCN+15 μM ZnCl2/33 μl NaCl;

RTA 2: 15 μM ZnCl2/33 μl NaCl.

At D18, the mice were sacrificed and the weight of the RTA and LTA muscles was measured. The results are given in the following table:

Muscle Weight (mg) Growth Mouse 4 RTA 4 53.6 8.77 LTA 4 58.3 Mouse 5 RTA 5 39.2 19.39 LTA 5 46.8 Mouse 6 RTA 6 40.1 24.69 LTA 6 50

These results show that an effect on muscle growth is indeed maintained in the presence of just this part of decorin.

Similar results were obtained with an even shorter peptide of 30 amino acids, with the sequence SEQ ID NO:15. 

1. Composition containing a fragment of decorin able to bind zinc to treat diseases associated with muscle wasting.
 2. Composition according to claim 1, characterised in that the diseases are selected from the group of neuromuscular diseases, to advantage muscular dystrophies such as Duchenne muscular dystrophy, and the cachexia.
 3. Composition according to claim 1 or 2, characterised in that the fragment comprises the sequence SEQ ID NO: 7 or
 15. 4. Composition according to claim 1 or 2, characterised in that the fragment has the sequence SEQ ID NO: 7 or
 15. 5. Composition containing decorin to increase muscle mass.
 6. Composition according to claim 5, characterised in that the aim of increasing muscle mass is to compensate for wasting resulting from immobilisation or old age.
 7. Composition according to claim 5 or 6, characterised in that it is for use in animals.
 8. Composition according to one of claim 5 or 6, characterised in that decorin is in the form of an active fragment.
 9. Composition according to claim 8, characterised in that the active fragment is able to bind zinc.
 10. Composition according to claim 9, characterised in that the fragment comprises the sequence SEQ ID NO: 7 or
 15. 11. Composition according to claim 9, characterised by the fragment has the sequence SEQ ID NO: 7 or
 15. 12. Composition according to claim 1 or 5, characterised in that the decorin is in the form of an active fragment, a recombinant protein, a fusion protein or a nucleic acid encoding such a protein or fragment.
 13. Composition according to claim 1 or 5, characterised in that the decorin is for intramuscular, intraperitoneal or intravenous injection.
 14. Composition according to claim 1 or 5, characterised in that the decorin is associated with other treatments, particularly gene therapy and cell grafting. 